Phase I/II study of the LAG-3 inhibitor ieramilimab (LAG525) ± anti-PD-1 spartalizumab (PDR001) in patients with advanced malignancies

Background Lymphocyte-activation gene 3 (LAG-3) is an inhibitory immunoreceptor that negatively regulates T-cell activation. This paper presents preclinical characterization of the LAG-3 inhibitor, ieramilimab (LAG525), and phase I data for the treatment of patients with advanced/metastatic solid tumors with ieramilimab ±the anti-programmed cell death-1 antibody, spartalizumab. Methods Eligible patients had advanced/metastatic solid tumors and progressed after, or were unsuitable for, standard-of-care therapy, including checkpoint inhibitors in some cases. Patients received ieramilimab ±spartalizumab across various dose-escalation schedules. The primary objective was to assess the maximum tolerated dose (MTD) or recommended phase II dose (RP2D). Results In total, 255 patients were allocated to single-agent ieramilimab (n=134) and combination (n=121) treatment arms. The majority (98%) had received prior antineoplastic therapy (median, 3). Four patients experienced dose-limiting toxicities in each treatment arm across various dosing cohorts. No MTD was reached. The RP2D on a 3-week schedule was declared as 400 mg ieramilimab plus 300 mg spartalizumab and, on a 4-week schedule (once every 4 weeks; Q4W), as 800 mg ieramilimab plus 400 mg spartalizumab; tumor target (LAG-3) suppression with 600 mg ieramilimab Q4W was predicted to be similar to the Q4W, RP2D schedule. Treatment-related adverse events (TRAEs) occurred in 75 (56%) and 84 (69%) patients in the single-agent and combination arms, respectively. Most common TRAEs were fatigue, gastrointestinal, and skin disorders, and were of mild severity; seven patients experienced at least one treatment-related serious adverse event in the single-agent (5%) and combination group (5.8%). Antitumor activity was observed in the combination arm, with 3 (2%) complete responses and 10 (8%) partial responses in a mixed population of tumor types. In the combination arm, eight patients (6.6%) experienced stable disease for 6 months or longer versus six patients (4.5%) in the single-agent arm. Responding patients trended towards having higher levels of immune gene expression, including CD8 and LAG3, in tumor tissue at baseline. Conclusions Ieramilimab was well tolerated as monotherapy and in combination with spartalizumab. The toxicity profile of ieramilimab in combination with spartalizumab was comparable to that of spartalizumab alone. Modest antitumor activity was seen with combination treatment. Trial registration number NCT02460224.


Sustained
Open access expression of these immune cell checkpoints can alter immune responses and contribute to T-cell suppression and subsequent immune dysfunction. 1 9 Dysregulation of immune checkpoints is a key mechanism by which tumors evade immune surveillance. 9 Blockade of LAG-3 has been shown to improve cytotoxic T-lymphocyte proliferation and effector function in vivo. 10 11 In addition, independent of MHC-II, LAG-3 has been shown to associate with the liver-secreted protein, FGL-1. 8 Blockade of the FGL-1-LAG-3 interaction by monoclonal antibodies (mAbs) suppressed tumor growth in established mouse models, in a receptor-ligand interdependent manner. 8 Data from syngeneic mouse models demonstrated that dual LAG-3/PD-1 blockade reduced tumor growth by increasing the proportion of effector T cells in the tumor. 12 A number of LAG-3-targeting molecules are currently in early stages of clinical development, with early results suggesting a modest benefit of single-agent, anti-LAG-3 treatment, supporting the potential of combination approaches. 13 Ieramilimab (LAG525) is a humanized immunoglobulin 4 (IgG4) (S228P) mAb that binds to LAG-3, resulting in inhibition of LAG-3 interaction with MHC-II molecules. Spartalizumab is a humanized IgG4 anti-PD-1 (S228P) mAb, which binds to PD-1 and blocks the interaction between the receptor and its ligands, programmed death-ligand 1 (PD-L1), and programmed death-ligand 2 (PD-L2). 14 Spartalizumab has shown clinical efficacy in various malignancies, including non-small cell lung cancer (NSCLC), 15 melanoma, 15 anaplastic thyroid cancer, 16 neuroendocrine neoplasms, 17 and nasopharyngeal cancer. 18 In this report, we present the preclinical characterization of ieramilimab and clinical data from a phase I study investigating ieramilimab as both a single agent and in combination with spartalizumab for the treatment of patients with advanced/metastatic solid tumors.

METHODS Preclinical characterization of ieramilimab
Ieramilimab is a humanized IgG4 antibody that contains the S228 hinge-stabilizing mutation and blocks the LAG-3-MHC-II interaction with low nanomolar affinity (data not shown). A plate-based Meso Scale Discovery (MSD) assay was developed to determine the ability of ieramilimab to neutralize the interaction between platebound FGL-1-His protein and biotinylated LAG-3-Fc protein. To establish the role of ieramilimab in enhancing cytokine secretion, naive B cells and T follicular helper (Tfh) cells were isolated from healthy human donor peripheral blood mononuclear cells and activated with Staphylococcal enterotoxin B (SEB) in the presence of ieramilimab or human IgG4 isotype control; supernatants were harvested, and cytokines were measured by MSD. The crystal structure of a human LAG-3 (first immunoglobulin variable domain (D1)) bound to the antigenbinding fragment of a humanized anti-LAG-3 antibody, ieramilimab, was determined. Detailed preclinical methods for in vitro assays and X-ray crystallography can be found in the online supplemental file (online only).

Study oversight
This study was performed in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by an independent ethics committee or Institutional Review Board at each study center. All patients provided written informed consent before any study procedures. The study was sponsored by Novartis Pharmaceuticals Corporation, which provided the study drug and worked with the investigators to design the study, collect, analyze, and interpret data.

Clinical study design
This phase I/II, open-label, multicenter study investigated the safety and efficacy of single-agent ieramilimab and in combination with spartalizumab in patients with advanced solid malignancies. Phase I consisted of two, staggered, dose-escalation arms: single-agent ieramilimab followed by ieramilimab in combination with spartalizumab.
Following completion of phase I, phase II was conducted in selected cancer indications.
Here, we present the data from phase I; data cut-off June 1, 2020.

Study objectives
The primary objective of phase I was to estimate the recommended phase II dose (RP2D) or maximum tolerated dose (MTD) of both single-agent ieramilimab and ieramilimab in combination with spartalizumab. Key secondary objectives included characterization of the safety and tolerability of single-agent ieramilimab and ieramilimab in combination with spartalizumab, assessment of pharmacokinetics (PK), and evaluation of preliminary antitumor activity. Biomarker analysis of pharmacodynamic effects was a key exploratory objective.

Patient population
Eligible patients for phase I were adults (≥18 years) with advanced/metastatic solid tumors who had either progressed on, were intolerant to, or were unsuitable for standard therapy, with an Eastern Cooperative Oncology Group (ECOG) performance status ≤2. Where feasible, patients were required to provide a new tumor biopsy at baseline and during treatment.
Key exclusion criteria were presence of symptomatic central nervous system (CNS) metastases or CNS metastases requiring local surgery; clinically significant cardiac disease or impairment; autoimmune disease; history of, or current, drug-induced pneumonitis; and systemic treatment with immunosuppressive medication, which could interfere with the study drugs, other than replacementdose corticosteroids in the setting of adrenal insufficiency.

Drug administration
Ieramilimab and spartalizumab were administered separately via intravenous infusions over 30 min, with at least Open access a 30-min break between administration of the two antibodies. Infusions for each antibody could be extended to up to 2 hours if clinically indicated, and the break between ieramilimab and spartalizumab infusions could be extended to up to 4 hours if clinically indicated. Ieramilimab was given first, followed by spartalizumab.
Treatment continued until unacceptable toxicity, progressive disease (PD) as per immune-related response criteria (irRC), 19 or patient/physician decision; guidelines are provided in the online supplemental file (online only). Treatment was also discontinued if consecutive doses (≥2) were missed due to drug-related toxicities; study treatment could be continued beyond disease progression for clinical benefit.
Dose-escalation decisions were based on all available safety, dose-limiting toxicity (DLT), PK, and pharmacodynamic data, and were guided by a Bayesian hierarchical logistic regression model following the escalation with overdose control principle. Dose escalation occurred until the MTD or RP2D was determined.

Safety assessments
Safety assessments included incidence and severity of adverse events (AEs) and serious AEs (SAEs), changes in laboratory values, physical examination, vital signs, ECOG performance status, and cardiac assessments. AEs were defined by the National Cancer Institute Common Terminology Criteria for Adverse Events V.4.03 and assessed at every visit. A DLT was defined as an AE of grade ≥3, suspected to be related to the study drug. The window for DLTs was one cycle for single-agent ieramilimab (eg, 28 days for Q4W and Q2W) and two cycles for ieramilimab and spartalizumab combination (eg, 56 days for a Q4W schedule and 42 days for a Q3W schedule).

Response assessments
Efficacy was evaluated by local investigator assessment per Response Evaluation Criteria In Solid Tumors (RECIST) V.1.1 and irRC. Tumor assessments were performed at screening (maximum 21 days before start of treatment); every 8 weeks (±1 week) after cycle 1, day 1 until 40 weeks, and then every 12 weeks (±1 week) until disease progression per irRC, or withdrawal from the study.

Assessment of PK
Blood samples for PK assessments were collected on days 1, 2, 8, 11, and 15 in cycles 1 and 3; day 1 in cycles 2, 4, 5, and 6; and at the end of treatment. Serum concentrations were determined with liquid chromatography mass spectrometry.

Biomarker assessments
Biopsy samples were collected at screening/baseline and between cycle 3 days 1-15; some on-treatment samples were provided during cycle 2, prior to a protocol amendment aligning samples with preclinical evidence on the timing of immune response to PD-1 blockade. Archival tumor samples were used for biomarker assessments in a limited number of cases.
For baseline and on-treatment samples, immune marker expression was assessed by immunohistochemistry (IHC) and gene expression by RNA-based analysis (further details can be found in the online supplemental file, online only).

Statistical methods
To declare the MTD, the following thresholds needed to be met: at least 6 patients treated at a given dose and a minimum of 21 patients for the single-agent arm of the trial or 15 patients for the combination arm. This given dose was recommended following review of all clinical data by Novartis and investigators.
Preclinical methodology is described in the online supplemental material (online only).

Preclinical characterization of ieramilimab
Ieramilimab demonstrated binding to D1 of LAG-3 through several continuous and discontinuous sequences covering the BC and DE loops, as well as the arginylglycylaspartic acid motif ( figure 1A,B). The recently described FGL-1-LAG-3 interaction has been reported to occur within D1 and D2 of LAG-3, independent of the MHC-II-LAG-3 interaction. 8 Using a novel MSD assay, we determined that ieramilimab blocked the LAG-3-FGL-1 interaction with a half-maximal inhibitory concentration (IC 50 ) of approximately 0.1 nM ( figure 1C). In three out of eight healthy donors tested, in a co-culture of SEBstimulated Tfh cells and B cells (online supplemental methods, online only), interferon gamma (IFN-γ) secretion was increased by blockade of LAG-3 with ieramilimab, relative to IgG control (figure 1D), demonstrating a Open access functional ability of ieramilimab to enhance a T-cell response.

Safety
AEs, regardless of study drug relationship, were observed in 132 (98.5%) and 120 (99.2%) patients in the singleagent and combination groups, respectively, and were comparable between treatment arms.
SAEs, regardless of study drug relationship and of any grade, were reported in 52 (38.8%) patients and 59 (48.8%) patients in the single-agent and combination groups, respectively. In the single-agent group, seven (5.2%) patients experienced at least one treatmentrelated SAE (TRSAE); the most common (≥1 patient) TRSAEs were vomiting (n=3, 2.2%) and diarrhea (n=2, 1.5%). Six (4.5%) patients had a fatal SAE, one (0.7%) of which, acute kidney injury, was considered treatment related; this patient experienced acute kidney injury secondary to worsening extensive tumor burden with histologic tumor necrosis consistent with grade 4 tumor lysis syndrome. In the combination group, seven (5.8%) patients experienced at least one TRSAE. Seven (5.8%) patients experienced a fatal SAE, none of which were treatment related.

PK of ieramilimab as single agent and ieramilimab in combination with spartalizumab
For both treatment groups, following ieramilimab treatment infusion, approximately dose-proportional increases in ieramilimab exposure (cycle 1 area under the plasma concentration-time curve (AUC tau )) were observed from 1 mg/kg to 15 mg/kg, as suggested by an approximate 20-fold increase in exposure with a 15-fold increase in dose (figure 3A,B; online supplemental table A2; online only).
Based on single-agent and combination dosing regimen data (Q2W, Q3W, and Q4W), exposure (eg, maximum concentration (C max ) or AUC tau ) during cycle 3 was higher compared with cycle 1, indicating moderate accumulation of ieramilimab. PK variability was low-to-moderate, as illustrated by between-subject variability (CV%), including a C max of 13.8%-34.6% and an AUC tau of 17.3%-45.6% at cycle 1 day 1 (N>3). The observed median effective halflife accounting for drug accumulation (T 1/2,eff ) of ieramilimab at cycle 3 ranged from 10 to 23 days.
The PK of spartalizumab in combination with different dose levels of ieramilimab were similar to the single-agent spartalizumab data at the same dose levels from a phase I study. 14

DISCUSSION
Immune checkpoint blockade with anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and/or anti-PD-(L)1 antibodies has transformed the treatment of several cancers, including melanoma and NSCLC, with improvements in overall survival. 23 Many patients are, however, unresponsive to existing checkpoint inhibitors or develop resistance during treatment, underscoring the need for Open access novel immunomodulatory approaches. 24 Key immunemediated mechanisms of resistance to checkpoint inhibitors include T-cell dysfunction, marked by the enhanced expression of co-inhibitory receptors; decreased T-cell priming and infiltration in the tumor microenvironment; suppression mediated by Tregs, myeloid-derived suppressor cells, and soluble factors; and loss of neoantigens/decreased antigen presentation. LAG-3 is an inhibitory receptor that is expressed in immune cells and has been shown, with PD-(L)1, to regulate T-cell exhaustion and inhibit an antitumor immune response. 5 Compensatory upregulation of LAG-3 has been related to adaptive resistance to immune checkpoint blockade, 25 supporting the hypothesis that targeting LAG-3 may be a promising therapeutic strategy to overcome immune checkpoint blockade resistance and improve patient outcomes. This first-in-human, dose-escalation trial demonstrated that ieramilimab is well tolerated, both as a single agent and in combination with spartalizumab. Low-grade fatigue, gastrointestinal side effects, pruritus, and fever were among the most commonly occurring TRAEs associated with single-agent ieramilimab use. There was no increase in incidence of immune-mediated SAEs, consistent with the observation that LAG-3 deficiency alone does not result in autoimmunity in preclinical models. 26 In contrast to combination checkpoint blockade with Figure 4 Duration of exposure and response plots. (A) Duration of exposure in patients receiving single-agent ieramilimab with best overall response of SD or NCRNPD, (B) Duration of exposure in patients receiving combination ieramilimab +spartalizumab with best overall response of CR, PR or SD, (C) Duration of response in patients receiving combination ieramilimab +spartalizumab with a best overall response of CR and PR. CR, complete response; NCRNPD, non-complete response/non-progressive disease (the presence of any non-target lesions or abnormal nodal lesions); PD, progressive disease; PR, partial response; SD, stable disease; UNK, unknown.

Open access
anti-CTLA-4 and anti-PD-1 agents, the immune-mediated toxicity of ieramilimab in combination with spartalizumab was comparable to that seen with spartalizumab alone. 14 No new safety signals were identified compared with existing immune checkpoint inhibitor treatments.
Ieramilimab demonstrated approximately doseproportional increases in exposure between the dose range of 1-15 mg/kg. Exposure of ieramilimab in combination with spartalizumab was within the range of exposure for both single-agent ieramilimab and spartalizumab, indicating no apparent drug-drug interaction between the two. Since there was no observed exposure response for safety or efficacy, and no MTD was reached, a target engagement receptor occupancy model was used to determine the RP2D, with the criteria of achieving 90% target engagement in >90% of patients. Similar approaches have been used to guide dosing of atezolizumab 27 and sabatolimab. 28 During dose escalation in a mixed population of advanced solid tumors, some of which had received prior treatment with checkpoint inhibitors, antitumor activity of single-agent ieramilimab was limited, consistent with preclinical models. 12 In contrast, ieramilimab and spartalizumab combination treatment was associated with SD or tumor shrinkage, including three CRs by RECIST and an additional CR by irRC in a patient with cervical cancer (online supplemental table A3, online only). While most PRs occurred in patients with tumor types known to respond to anti-PD-1 antibodies, antitumor activity was observed in several tumor types where previous effectiveness of immunotherapy has not been established in a consistent way, including adrenocortical carcinoma and PD-L1-negative TNBC (online supplemental figure A1,2). 29 In addition, the duration of response has exceeded 4 years in some patients, suggesting that long-term combination therapy is tolerable (figure 4B) and potentially augmented by LAG-3 blockade. For both ieramilimab doses at 400 mg Q3W and 800 mg Q4W, over 90% of patients were predicted to have at least 90% target engagement. At the alternative dosing regimen of ieramilimab 600 mg Q4W, 90% of patients were predicted to have at least 88% target engagement. This, therefore, indicates a comparable target engagement with ieramilimab doses at 600 mg Q4W and 800 mg Q4W.

Open access
In vitro, ieramilimab blocks the interaction between LAG-3 and both MHC-II and FGL-1, with high affinity. Elevated levels of FGL-1 in cancer may contribute to suppression of activated T cells and evasion of antitumor immunity, 8 however, relative contributions of disrupting LAG-3 interactions with FGL-1 or MHC-II in patients is unclear. Although not addressed in this study, further translational investigation is warranted.
A large number of baseline tumor samples were collected to explore pharmacodynamic effects and potential efficacy predictors of ieramilimab, as both a single agent or in combination with spartalizumab. IHC and RNA sequencing analyses suggested that tumor stability or response following combination treatment was associated with baseline immune-inflamed gene expression patterns similar to the IFN-γ signature associated with response to the PD-1 inhibitor, pembrolizumab. 30 In patients who received single-agent ieramilimab treatment, baseline T-cell inflamed signatures tended to be higher in tumor samples from those who exhibited SD (online supplemental figure A4C, online only). Among the heterogeneous tumors enrolled during the dose-escalation portion of the study, LAG-3 expression, per se, was not a predictive biomarker, except insofar as LAG-3 correlated with immune-inflamed gene expression patterns overall.
Consistent with the above observations regarding baseline immune gene expression, on-treatment biopsies suggested that patients with tumors that responded to ieramilimab in combination with spartalizumab demonstrated upregulation of already high baseline CD8 or T-cell inflamed expression levels. In several cases, however, tumor reduction occurred in the context of relatively immune-cold profiles at baseline, where on-treatment biopsies demonstrated increased levels of CD8 and PD-L1 following ieramilimab and spartalizumab treatment. The relative impact of ieramilimab on this effect is unknown and limited by the small number of PRs in this mixed group of tumor indications, as well as the smaller number of available on-treatment biopsies.
Despite preclinical models demonstrating synergistic antitumor activity with LAG-3 and PD-1 co-blockade, 12 the modest antitumor activity observed in this clinical trial in a multitumor, unselected patient population, highlights the challenges in developing next-generation combination immunotherapies. Although the relative contribution of ieramilimab to antitumor efficacy could not be determined clinically or through translational analyses conducted in this study, a subset of patients experienced long-term clinical benefit with ieramilimab and spartalizumab. Consistent with a potential contribution of LAG-3 targeting, previous data on the combination of the LAG-3 inhibitor, relatlimab, with the PD-1 inhibitor, nivolumab, in patients with melanoma who had received prior immunotherapy, showed objective response rates of approximately 12%, with a disease control rate of 49% for the doublet. 31 32 In the phase III RELATIVITY-047 study, relatlimab, in combination with nivolumab, demonstrated statistically significant progression-free survival benefit (10.1 months (95% CI: 6.4 to 15.7)) compared with nivolumab monotherapy (4.6 months (95% CI: 3.4 to 5.6), HR, 0.75 (95% CI: 0.6 to 0.9); p=0.0055) in patients with previously untreated metastatic or unresectable melanoma; this difference was likely driven by the LAG-3 positive (≥1%) subgroup. 33 These results further highlight the clinical potential of dual LAG-3/ PD-1 inhibition. Our phase I study showed responses to the dual anti-LAG-3/anti-PD-1 therapy in patients with various cancer indications, including confirmed CRs per RECIST V.1.1, in three patients with thymoma, adrenocortical carcinoma, and triple-negative breast cancer, as well as an additional CR by irRC in a patient with cervical cancer. Furthermore, the possible contribution of anti-LAG-3 to the durability of combination therapy response is supported by seven patients who received ieramilimab plus spartalizumab for over 3 years, including two of the patients achieving CR, one patient with a CR by irRC, plus four additional patients with mesothelioma, nasopharyngeal cancer, gastric cancer, and a malignant neoplasm of unknown primary who achieved PR. These data suggest that LAG-3 targeting may contribute to anti-PD-1 activity in different cancers beyond melanoma. Consistent with this, in the phase II part of our study, ieramilimab in combination with spartalizumab elicited durable responses not only in melanoma, but also in patients with mesothelioma and renal-cell carcinoma who had received prior treatment with anti-PD-(L)1 inhibitors. 34 The clinical impact of targeting LAG-3 in combination with other immunotherapies warrants further investigation.

Patient consent for publication Not required.
Ethics approval This study was performed in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by an independent ethics committee or institutional review board at each study center. All patients provided written informed consent before any study procedures. The study was sponsored by Novartis Pharmaceuticals Corporation which provided the study drug and worked with the investigators to design the study, collect, analyze, and interpret data. Participants gave informed consent to participate in the study before taking part.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available upon reasonable request.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.
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